ERAP1 enzyme-mediated trimming and structural analyses of MHC I-bound precursor peptides yield novel insights into antigen processing and presentation

Abstract

Endoplasmic reticulum aminopeptidase 1 (ERAP1) and ERAP2 critically shape the major histocompatibility complex I (MHC I) immunopeptidome. The ERAPs remove N-terminal residues from antigenic precursor peptides and generate optimal-length peptides (i.e. 8-10-mers) to fit into the MHC class I groove. It is therefore intriguing that MHC class I molecules can present N-terminally extended peptides on the cell surface that can elicit CD8+ T-cell responses. This observation likely reflects gaps in our understanding of how antigens are processed by the ERAP enzymes. To better understand ERAPs' function in antigen processing, here we generated a nested set of N-terminally extended 10-20-mer peptides (RA) _n AAKKKYCL covalently bound to the human leukocyte antigen (HLA)-B*0801. We used X-ray crystallography, thermostability assessments, and an ERAP1-trimming assay to characterize these complexes. The X-ray structures determined at 1.40-1.65 Å resolutions revealed that the residue extensions (RA) _n unexpectedly protrude out of the A pocket of HLA-B*0801, whereas the AAKKKYCL core of all peptides adopts similar, bound conformations. HLA-B*0801 residue 62 was critical to open the A pocket. We also show that HLA-B*0801 and antigenic precursor peptides form stable complexes. Finally, ERAP1-mediated trimming of the MHC I-bound peptides required a minimal length of 14 amino acids. We propose a mechanistic model explaining how ERAP1-mediated trimming of MHC I-bound peptides in cells can generate peptides of canonical as well as noncanonical lengths that still serve as stable MHC I ligands. Our results provide a framework to better understand how the ERAP enzymes influence the MHC I immunopeptidome.

Keywords: CD8+ T cells; HLA-B*0801; adaptive immunity; antigen presentation; antigen processing; endoplasmic reticulum (ER); endoplasmic reticulum aminopeptidase (ERAP); immunology; immunopeptidome; major histocompatibility complex I (MHC I); structural biology.

Figure 1.

Presentation of 10-mer (R(N-Me)A)AAKKKYCL by HLA-B*0801E76C. A, top, molecular surface of HLA-B*0801E76C groove (light gray) showing the bound (R(N-Me)A)AAKKKYCL (green) with P−1 (N-Me)Ala and P−2 Arg (see B) extending out of the A pocket. Peptide residue positions (P), A pocket, and α-helices are indicated. Bottom, bound conformation of (R(N-Me)A)AAKKKYCL (green) shown against the α1-helix of HLA-B*0801E76C (light gray); the AAKKKYCL core binds into the groove, whereas P−1 (N-Me)Ala and P−2 Arg (see B) protrude out. The disulfide bond (yellow) between P7 Cys and Cys⁷⁶ is shown. Peptide residues are labeled. B, 2mF_o-DF_c electron density, contoured at 1σ, is shown as blue mesh around (R(N-Me)A)AAKKKYCL. The density is visible for all residues except for the N-methyl group of P−1 (N-Me)Ala and P−2 Arg. C, superimposition of the canonical structure of HLA-B*0801–bound GGKKKYKL (orange) (PDB entry 1AGD) with the structure of HLA-B*0801E76C–bound (R(N-Me)A)AAKKKYCL (green), shown against the α1-helix of HLA-B*0801E76C (light gray). D, same as in C, showing that in the canonical structure of HLA-B*0801–bound GGKKKYKL (orange), Arg⁶² occupies a position that blocks the A pocket. In contrast, in the structure of HLA-B*0801E76C-bound (R(N-Me)A)AAKKKYCL (green), Arg⁶² has moved up and out, which opens the A pocket and allows the extensions to protrude out. E, details of interactions within the A pocket showing rotation of P1 Ala and its effect on P−1 (N-Me)Ala position. The H-bonds between the main-chain nitrogen of P1 Ala and Asn⁶³ and between the main-chain carbonyl oxygen of P1 Ala and Tyr¹⁵⁹ are shown as green dashed lines.

Figure 2.

Binding of a nested set of N-terminally extended peptides (R(N-Me)A)(RA)_n-1AAKKKYCL into HLA-B*0801E76C. A, superimposition of the structures of 10-mer (green), 12-mer (cyan), 14-mer (magenta), and 20-mer (yellow) (R(N-Me)A)(RA)_n-1AAKKKYCL, shown against the α1-helix of HLA-B*0801E76C/10-mer complex (light gray). The backbone conformations of the peptides are very similar except between P1 and P3. B, superimposition of the structures of the 10-mer (green) and 12-mer (cyan) (R(N-Me)A)(RA)_n-1AAKKKYCL, shown within the A pocket of HLA-B*0801E76C/10-mer complex (light gray). A water molecule (cyan) occupies the A pocket in the 12-mer structure. Hydrogen bonds are shown as green and dark blue dashed lines for the 10- and 12-mer, respectively. C, same as in A, showing that in the canonical structure of HLA-B*0801-bound GGKKKYKL (orange), the configuration of Arg⁶² “closes” the A pocket. Arg⁶² changes its position significantly upon binding the 10-mer (green) (see also Fig. 1D), 12-mer (cyan), 14-mer (magenta), and 20-mer (yellow).

Figure 3.

Examining the effect of P−5 (N-Me)Ala and P5 middle anchor residues. Superimposition of the HLA-B*0801E76C–bound 14-mers (RA)₃AAKKGYCL (lime) and (R(N-Me)A)(RA)₂AAKKKYCL (magenta). The two peptides bind in a nearly identical manner and share very similar structural features. The peptide residue positions (P) are indicated.

Figure 4.

Thermal denaturation assay. A, averaged thermal denaturation curves for individual peptides, as indicated, presented by HLA-B*0801E76C. B, graphs of the first derivative of the curves shown in A. The T_m values, determined from the minima, are indicated for each peptide. The T_m value of each complex was unchanged with different refolding batches.

Figure 5.

ERAP1-mediated trimming of free 14-mer (RA)₃AAKKKYCL and 20-mer (RA)₆AAKKKYCL. Free peptides (∼12 μg) were incubated with 0.4 μg of ERAP1 at 37 °C. An aliquot was taken after 20 min (14-mer) and 30 min (20-mer) and analyzed by MS. The starting peptides and their fragments are indicated.

Figure 6.

ERAP1-mediated trimming of HLA-B*0801E76C-bound peptides. MHC I–bound 14-mer (RA)₃AAKKKYCL (A) and 20-mer (RA)₆AAKKKYCL (∼12 μg of peptide) (B) were incubated with ∼34 μg of ERAP1 at 37 °C. Aliquots were taken from the mixtures at the indicated times and analyzed by MS. Additional ERAP1 was added to the mixtures after the aliquots of 3.5 and 6 h. The starting MHC I–bound peptides and fragments are indicated.

Figure 7.

Trimming of MHC I–bound 18-mer precursor peptide by the ERAP enzymes. Shown is a model of antigen processing in which an N-terminally extended candidate peptide is bound into the MHC I groove by only a few C-terminal residues. As the peptide undergoes a dynamic binding and “sampling” into the groove (indicated by red arrows), from its C to N terminus, the N-terminal residue extensions are concurrently trimmed by the ERAPs. Inside the cells, the ERAP1 and ERAP2 enzymes likely exist in more than one molecular form, with each form shaping differently the MHC I immunopeptidome (see “Discussion”). ERAP1/ERAP2 indicates the heterodimer.